Successes and limitations of quantitative diet metabarcoding in a small, herbivorous mammal

DNA metabarcoding is widely used to determine wild animal diets, but whether this technique provides accurate, quantitative measurements is still under debate. To test our ability to accurately estimate the abundance of dietary items using metabarcoding, we fed wild-caught desert woodrats (Neotoma lepida) diets consisting of constant amounts of juniper (Juniperus osteosperma, 15%) and varying amounts of creosote (Larrea tridentata, 1%-60%), cactus (Opuntia sp., 0%-100%) and commercial chow (0%-85%). Using metabarcoding, we compared the representation of items in the original diet samples to that in the faecal samples to test the sensitivity and accuracy of diet metabarcoding, the performance of different bioinformatic pipelines and our ability to correct sequence counts. Metabarcoding, using standard trnL primers, detected creosote, juniper and chow. Different pipelines for assigning taxonomy performed similarly. While creosote was detectable at dietary proportions as low as 1%, we failed to detect cactus in most samples, probably due to a primer mismatch. Creosote read counts increased as its proportion in the diet increased, and we could differentiate when creosote was a minor and major component of the diet. However, we found that estimates of juniper and creosote varied. Using previously suggested methods to correct these errors did not improve accuracy estimates of creosote, but did reduce error for juniper and chow. Our results indicate that metabarcoding can provide quantitative information on dietary composition, but may be limited. We suggest that researchers use caution when quantitatively interpreting diet metabarcoding results unless they first experimentally determine the extent of possible biases.